Acute effects of oral mesna administration on the full amino acid profile and 3-methylhistidine: secondary results from the CYLOB dose-finding study

Plasma total cysteine (tCys) is strongly associated with fat mass in humans. Mesna lowers plasma tCys in a dose-dependent manner, but it is not known whether it interferes with metabolism of other amino acids or protein. In this Phase-1 study, we show that a single dose of mesna administered at 400, 800, 1200 or 1600 mg to 6–7 individuals per dose only slightly affects amino acid profiles, with increases in plasma valine across dose levels. There were no effects of mesna on 3-methylhistidine, a marker of protein breakdown. Supplementary Information The online version contains supplementary material available at 10.1007/s00726-024-03398-2.


Introduction
Plasma concentrations of total cysteine (tCys) are strongly and linearly associated with overweight and obesity (Elshorbagy et al. 2008(Elshorbagy et al. , 2012)).Dietary restriction of cysteine and its precursor, methionine, leads to significant restriction may therefore contribute to the observations of lower lean, bone and skeletal muscle mass as compared to controls (Ables et al. 2012;Cooke and Ables 2023;Cooke et al. 2020).
As opposed to dietary cysteine restriction, pharmacological reduction of cysteine using mesna reduce fat mass accumulation but not lean mass accumulation in mice (Vinknes et al. 2023).Taken together with previous evidence (Martinez et al. 2022;Nichenametla et al. 2018;Nichenametla et al. 2022), these demonstrations indicate that the amino acid pool available for protein synthesis may be affected differently in pharmacologic compared to dietary manipulation of cysteine.However, there is no evidence on the effect of mesna on the plasma amino acid pool and/or protein breakdown in humans.To explore this, we utilized data from our previous dose-escalation trial with mesna in humans with overweight and obesity, and investigated whether a single oral dose of mesna (400, 800, 1200 and 1600 mg/d) influenced plasma and urinary excretion of 20 amino acids and 3-methylhistidine, a marker of protein breakdown.

Methods
The present data are from a phase-1, non-randomized single ascending dose study in healthy volunteer men with overweight and obesity.Details on the study protocol, study design, blood sampling, and main results have been published elsewhere (Vinknes et al. 2023).Briefly, 17 men were assigned to receive 400 mg (n = 7), 800 mg (n = 6), 1200 mg (n = 6) and 1600 mg (n = 6) of oral mesna.Blood samples were taken at baseline and 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10 and 12 h post-dose into EDTA-lined tubes.Twenty-four-hour urine was collected as described previously (Vinknes et al. 2023).Blood and urine were stored at − 80 ℃ until analysis.Plasma and urine amino acid profiles, and 3-methyl-histidine were measured using liquid chromatography-tandem mass spectrometry using previously published methods (Turner et al. 2023).Measured analytes included alanine, arginine, asparagine, aspartate (plasma only), glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, ornithine, phenylalanine, proline, serine, taurine, threonine, tryptophan, tyrosine and valine.All study participants gave written informed consent.The study was performed according to the Declaration of Helsinki, and approved by Regional Ethics Committee South-East (Ref no.87266).
Area under the plasma concentration versus time curve (AUC) was calculated for each amino acid using the trapezoidal method.P trend was calculated using linear regression in separate models for each amino acid, where the AUC was the dependent variable and the dose was the independent variable.P-values < 0.05 were considered statistically significant.

Baseline characteristics
Baseline characteristics of the participants has been published previously (Vinknes et al. 2023).Briefly, n = 17 men with a mean (SD) age of 43.5 (8.4) years and BMI of 32.7 (3.0) kg/m 2 participated in the present study.Baseline amino acid profiles according to dose level can be viewed in Supplemental Table S1.

Plasma and urinary excretion of amino acids by dose level
The AUC for valine increased significantly with increasing dose levels (β = 110 µmol×h/L, 95% CI: 14.6, 205, p trend = 0.03).Similar responses were observed for isoleucine and tryptophan, but these findings did not reach statistical significance (p trend < 0.10, Table 1).No effects of mesna were observed on plasma levels of 3-methylhistidine.Furthermore, no effects of escalating mesna dosing were observed on 24-h urinary excretion of amino acids or 3-methylhistidine (Supplemental Table S2).

Discussion
In this secondary analysis of a dose-escalation study, we report that administration of increasing single doses of mesna, which effectively lowers plasma tCys (Vinknes et al. 2023), only minimally influences the remaining plasma amino acid pool and 3-methylhistidine.These findings indicate that there are few acute effects of mesna on amino acid metabolism.However, a drawback of the present study is that we were not able to quantify tissue concentrations or flux.In rodents, dietary restriction of cysteine alters plasma concentrations of histidine, phenylalanine, serine, threonine and tryptophan possibly by directing them to the TCA cycle (Gao et al. 2019;Nichenametla et al. 2022), with concomitant lower rates of protein synthesis, reduced growth rates and lower lean mass (Ables et al. 2012;Cooke and Ables 2023;Cooke et al. 2020).Therefore, long-term studies with mesna administration are needed to investigate whether our reported minor effects on the plasma amino acid pool persist over time, and whether there are negative effects on lean mass.
Although we mostly observed neutral effects of a single oral dose of mesna across different dose levels, there 1 3 were indications of effects on branched-chain amino acids (BCAA).There was a significant increase in valine and a trend for an increase in isoleucine across dose levels.The underlying mechanism is unclear, but is likely indirect considering that mesna works by binding thiols in plasma and facilitate their urinary excretion.Speculatively, our results might relate to the effect of mesna on endogenous cysteine availability.Elimination of dietary cysteine rapidly induces autophagy in multiple experimental model systems (Cooke et al. 2020;Plummer and Johnson 2019;Ruckenstuhl et al. 2014) similar to short-term fasting (Shabkhizan et al. 2023).Notably, during 24 h of fasting in humans, plasma concentrations of BCAAs are elevated by ~ 14 to 38% with only negligible changes in other amino acids (Anfinsen et al. 2023).However, the study in question also reported the largest increase in plasma leucine, which we did not observe after ascending single doses of oral mesna.As such, we cannot exclude the possibility that the observed effect on valine and the weaker effect on isoleucine were due to chance.This is the first study reporting on the acute effects of mesna on amino acids and 3-methylhistidine in humans with overweight and obesity.It provides preliminary, yet valuable information on whether mesna can affect the metabolism of other amino acids.Our study has clear limitations, including the small sample size, inclusion of men only, the short study duration, and the lack of a control group.In addition, the study was not designed to detect effects of mesna on the plasma or urine amino acid profile.These limitations impede the interpretation and generalizability of our findings.Thus, the results of this study are therefore mainly suitable to inform the planning and design of future studies, where these limitations can be overcome.
In conclusion, the acute effects of mesna on plasma and urinary concentrations of amino acids and 3-methylhistidine are minimal, but experimental and long-term human studies are needed to address whether repeated mesna dosing over time affects amino acid metabolism.

Table 1
Mean (standard deviation) area under the curve of plasma amino acid concentrations according to mesna dosing